Many studies have confirmed that caloric restriction (CR) (synonymously, dietary restriction) extends lifespan in a range of non-human organisms including budding yeast (Saccharomyces cerevisiae), worms (Caenorhabditis elegans), the fruit fly (Drosophila melanogaster), and the mouse (Mus musculus). Based on the broad conservation of CR in animals, it is likely that a similar mechanism or mechanisms for CR-based lifespan extension also operates in humans. This intriguing observation opens the way to the possible extension of human lifespan by oral medications or other interventions, as opposed to (or as a supplement to) changes in diet, socioeconomic status, access to healthcare, etc.
In addition to the effects of CR on lifespan, other studies suggest that CR is likely to delay the onset or reduce the incidence of age-related diseases in humans, including cancer, diabetes, and cardiovascular disease, thus offering up a second critical reason to study the mechanism(s) of action of CR. Thus, for example resveratrol, a plant product that is a component of red wine, has been shown to have positive effects on the health and survival of “middle-aged” or overweight mice in ways that may correlate with protective methods for, e.g., diabetes (see, e.g., Baur et al., Nature (2006) 444:337-342), and has also been shown to provide protection against metabolic disease (see, e.g., Lagouge et al., Cell (2006) 127:1109-1122). In both cases, the action of resveratrol is thought to be mediated at least partially by some of the same mechanisms that are involved with CR-based lifespan extension, e.g., by the sirtuin family of genes which are thought to be involved in the CR-mediated lifespan extension response. Therefore, on this basis it is likely that an understanding of the basis or bases for CR action could result in treatments for these age related diseases in addition to methods of extending longevity.
On the basis of the above observed effects of CR on longevity and disease, considerable effort has been devoted to understanding the mechanism(s) of action of CR to produce these effects, for example by identifying the components of the CR pathway(s) by altering or mutating genes and screening for those gene alterations or mutations that change the CR response. One result of such studies has been the identification of the silent information regulator 2 (Sir2) family of protein deacetylases, also known as the sirtuins, which are found in a wide range of organisms ranging from bacteria to humans, and which have been shown to extend longevity in, e.g., yeast and the nematode worm. See, e.g., Bitterman et al., Microbiol. Mol. Biol. Rev. (2003) 67:376-399. However, other studies have shown that it is likely that there are other CR-based longevity pathways that act in parallel with those involving the sirtuins, offering up the possibility of additional pathways for interventions for increasing human longevity or reducing human disease. See, e.g., Kaeberlein et al., PLOS Biology (2004) 2:1381-1387 and Kaberlein et al., PLOS Biology (2007) 3:0655-0660 (available at plosgenetics.org); see also Medvedik et al., PLOS Biology (2007) 5:e261.
As an alternative to identifying the components of the CR pathway(s) by gene alteration or mutation, these components can also be characterized by identifying compounds that alter the CR response and then determining what molecules those compounds interact with. As noted above, although the sirtuins may be involved in the CR response, there is evidence to suggest that there are other CR-based longevity pathways, e.g., compounds unrelated to resveratrol that activate SIRT1 (identified by in vitro biochemical screen using purified SIRT1) and have important physiological effects in mice. SIRT1 is the mammalian homolog of the budding yeast silent information regulator 2 (SIR2), which encodes a histone deacetylase that has been implicated in the control of lifespan and the mitigation of age-associated diseases by CR regulatory mechanisms. See Milne et al., Nature (2007) 450:712-716. Identification of these pathways may be made by understanding the molecular effects of compounds identified as acting outside previously characterized pathways and, once identified, the components of these pathways may serve as new target molecules for modulating the CR response. See, e.g., Petrascheck et al., Nature (2007) 450:553-557.
Additionally, compound-based screens have another distinct advantage, in that the compounds identified by these screens have utility not simply for their usefulness in identifying the components of the CR pathway(s) but also because these compounds themselves, or in modified form, may be used as drugs for stimulating the CR response. Thus, for example, a compound shown in a particular model system (e.g., yeast, worms, fruit fly, mouse) to alter the CR response may be used directly, or in chemically modified form, to achieve the same result in monkeys and, ultimately, humans. The need for a variety of compounds altering the CR response is clear; resveratrol, for example, has low bioavailability and therefore is not necessarily a particularly suitable compound for altering the CR response.
One example of such a compound-based screen is the cell-based phenotypic “Death of Daughters” (DeaD) assay provided in U.S. patent application Ser. No. 10/790,456 to Goldfarb, the contents of which are herein incorporated in their entirety by reference. As described in this reference, the DeaD assay allows for the high throughput screening of compounds in yeast cells for those compounds that extend or shorten what is termed “replicative aging,” i.e., aging as defined as the number of divisions an individual yeast cell undergoes before dying. In yeast, because cell division is asymmetric, it is straightforward to distinguish a newly formed small “daughter” cell from the larger “mother” cell that gave rise to the daughter by division, and therefore it is possible to monitor the number of divisions a mother cell undergoes by distinguishing these cells from their progeny. Typically this discrimination is done by a trained microscopist, and, although straightforward, is extremely labor- and time-intensive. However, the DeaD assay makes use of yeast strains that have been genetically engineered so that daughter cells die, thereby allowing for replicative assays based on the growth properties of bulk populations of cells which, because the daughters die, are essentially mothers only, i.e., methods that are quick and require relatively little labor to perform, since they are based on bulk properties (absorbance) rather than on detailed microscopic analyses.
The high throughput screening of compounds in yeast cells performed with the DeaD assay may be done on yeast cells exposed to the test compounds only; alternatively, or in addition, the DeaD assay may be done with yeast cells also treated with an agent or agents that alter longevity or other aspects of the CR response, in order to identify test compounds which counter the effects of this agent or agents. For example, the Sir2 protein, like the other sirtuins, is a NAD+-dependent deacetylase which produces nicotinamide (also referred to herein as NIC or NAM) as a reaction product. Nicotinamide in turn acts as a non-competitive inhibitor of the Sir2 protein and Sir2-like enzymes in vitro and, in vivo, and can accelerate yeast ageing by inhibiting Sir2. see, e.g., Anderson et al., Nature (2003) 423:181-185. Therefore, in addition to using the DeaD assay to screen for compounds altering the CR response in untreated yeast cells, additional information on compounds altering the CR response can be obtained by using yeast cells treated with nicotinamide, i.e., in a situation where compounds are selected based on their ability to counter the longevity-shortening effects of nicotinamide.